Replacing animal model testing with ex vivo and in vitro testing
In vivo (from the Latin "in the living") is an experiment using a whole living organism as opposed to a partial or dead organism, or a controlled environment in vitro.
Animal experimentation and clinical trials are both forms of in vivo research.
In vivo tests are historically used in preference to in vitro tests because they are better suited to observing the overall effects of an experiment on a living subject and may be required in the regulatory framework.
Ex vivo (Latin: "outside the living") refers to what happens outside an organism.
In science, ex vivo refers to experimentation or measurements performed in or on tissues in an artificial environment outside the organism with minimal alteration of natural conditions.
A widely used ex vivo study is the chickchorioallantoid membrane test (CAM).
Other skin explant studies are commonly used in cosmetic research and development.
In vitro (Latin: in glass) studies in experimental biology are those conducted using components of an organism that have been isolated from their usual biological environment. Three-dimensional (3D) in vitro culture has developed greatly in recent years, particularly through the use of gel matrices. 3D culture is more representative of the in vivo environment, especially in cell-cell interactions. Complex in vitro reconstruction models exist, such as reconstructed epidermis models.
Why switching from in vivo to alternative models?
- Developing relevant alternative solutions - a panel of tests to answer today's animal-only questions
- Reduce the costs of complex ex vivo or in vitro models
- Preparing for the transition to the regulatory level
Cell lines: selection criteria
Cell lines are the basis of the research work of many laboratories.
Their choice is crucial and must be made in an informed manner, taking into account the following criteria.
Authenticity of the line
There are more than 400 misidentified cell lines registered in a specific database.
So it is important, when you start with a new cell line, to be sure it is what you think it is. Be sure to obtain your cell line from a trusted cell bank such as ATCC, DSMZ or ECACC.
Check for contamination
Despite the risk of erroneous or non-reproductive results associated with mycoplasma infection of a cell culture, they are often overlooked because of the size of the microorganism and their structural differences from other bacteria.
However, many tests exist to verify the absence of mycoplasma: PCR, ELISA, DNA labelling, growth tests, etc.
Malgré le risque de résultats erronés ou non reproductif lié à une infection d’une culture cellulaire par mycoplasme, elles sont souvent négligées, en raison de la taille du micro-organisme et de leurs différences structurelles par rapport aux autres bactéries.
De nombreux tests existent cependant pour vérifier l’absence de mycoplasme : PCR, ELISA, marquage ADN, tests de croissance, etc.
Prefer a low passage number
Cell lines have a certain genetic instability: at each division, errors become integrated into the genome of the cells, creating differences over time.
These differences are regularly revealed by comparing the profiles of equivalent primary and cultured cell lines that have undergone a high number of passages. Good practices include starting from a stock of corresponding low-pass cells on a regular basis (2-3 months, depending on the lineage), and referencing the estimated number of passes in each experiment.
For each biological question, a preferred cell line?
If the biological question can be related to a particular pathology, then it is essential to choose the cell line that will then become the "model" of the disease. However, the choice, however critical it may be, is not always obvious. As a starting point for research, the Cancer Cell Line Encyclopaedia (CCLE) provides public access to genetic data for about 1000 cell lines and helps guide choices.
The other reference source is the COSMIC (Catalogue of Somantic Mutations in Cancer) database, which also provides data on mutations in cancer cell lines.
If the biological question is independent of a particular pathology, then the reference strains are typically those used in previous research work.
However, this approach introduces a bias, since some lines are more used than others.
The appropriate scientific solution could be, for example, to test several lines on an already validated test, which reinforces the relevance of the observations made on a line used for the whole study.
Find our blog article on HeLa cells to learn more about this cell line with 75 000 scientific articles!
Types of providers
Multiple actors are involved in cell culture: service companies, cell suppliers, and specialized academic platforms.
Many service companies produce tools for cell culture such as explants, specific culture media. Specialized companies can also generate stable cell lines or induced pluripotent cells (IPSCs) and perform tests on them.
Primary lines and cell lines are typically provided by private or public cell banks.
Academic platforms specialized in cell biology offer in vitro tests, depending on their capabilities.
Why does a primary culture stop (or continue) growing?
Primary cell culture is the dissociation of cells from an animal or plant parental tissue by enzymatic or mechanical measures and the maintenance of cell growth in a suitable substrate, in glass or plastic containers, under controlled environmental conditions.
Cells derived from primary cultures have a limited lifespan. Cells cannot be kept indefinitely for several reasons. Increasing the number of cells in primary culture will cause depletion of substrate and nutrients. In addition, cell activity will gradually increase the level of toxic metabolites in the culture, which will inhibit cell growth.
The cells can then be resuspended and placed in a new medium, thus eliminating toxic metabolites. This is what constitutes a secondary culture, the goal is to generate a larger number of cells, and to keep the cultures alive.
However, there is a risk that the cells may evolve and transform or acquire genetic modifications. The quality of the test will be determined by the capacity for analysis and interpretation.